Splicing of
RPGR was analyzed in seven Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) derived from the blood of male patients with RP (
Table 1) and an unaffected male control. Informed consent was obtained from each patient and the unaffected control in this study, and all were treated in accordance with the Declaration of Helsinki. Additional family members were not available for further tests. LCLs were grown in RPMI 1640 medium (LabForce, Nunningen, Switzerland), 10% FBS (LabForce), 1.1% penicillin-streptomycin (LabForce), and 1.3% L-glutamine (LabForce) at 37°C, 5% CO
2 by using 75-cm
2 cell culture flasks (Techno Plastic Products, Trasadingen, Switzerland). Between 8 × 10
6 and 4 × 10
7 cells were pelleted by centrifugation at 4°C with 2000
g for 10 minutes. The pellets were washed with 1× DPBS (LabForce), snap frozen, and stored at −80°C before extraction of DNA or RNA. To inhibit nonsense-mediated mRNA decay (NMD), 1 × 10
6 cells/mL were incubated in 30 μg/mL cycloheximide (Sigma-Aldrich, Schnelldorf, Germany) at 37°C, 5% CO
2 in 25-cm
2 cell culture flasks (Techno Plastic Products) for 4 hours, as previously described.
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